Experimental autoimmune encephalomyelitis (EAE) is a CD4 T cell-mediated inflammatory CNS demyelinating disease that serves as a model for human multiple sclerosis (MS). Relapsing-remitting EAE (R-EAE) is induced in SJL/J mice following active immunization with encephalitogenic myelin peptides (e.g., PLP139-151 or MBP84-104) or by the adoptive transfer of peptide-specific T cells. Our published studies have shown that pathologic disease progression (relapses) is due to the recruitment of T cell responses against non-crossreactive endogenous myelin epitopes on the same or different myelin proteins (intramolecular or intermolecular epitope spreading). Delineating the cellular and molecular mechanisms underlying this process is critical for understanding the pathogenesis of human relapsing-remitting and chronic-progressive autoimmune diseases (e.g., MS, RA, and SLE). Building upon our recent work, we will continue to test the hypothesis that priming of T cells specific for endogenous myelin epitopes occurs primarily in the CMS itself and is driven by endogenous antigen presented by peripheral dendritic cells (DCs) which infiltrate the inflamed CMS. Aim 1 will use multiple assays (ELISPOT analysis, intracellular cytokine staining, and MHC class II tetramers) to precisely quantitate the temporal appearance of Th1/Th2 cells in peripheral lymphoid organs, peripheral blood and the CMS in mouse models of relapsing, chronic and acute EAE specific for a panel of potential encephalitogenic myelin epitopes, assess their T cell receptor (TCR) repertoire (using immunoscope), and analyze their encephalitogenic (using tolerance and adoptive transfer strategies) vs. regulatory potential. Highly sensitive in vivo transfer systems employing CFSE-loaded naive TCR transgenic T cells and bone marrow chimeras will be used to assess the temporal appearance, CMS homing requirements, and the anatomic location(s) of activation of naive myelin-specific T cells stimulated by endogenously released myelin epitopes, as well as test if antigen availability determines the hierarchy of T cell epitope spreading. Aim 2, with an emphasis on CNS-resident DCs, will delineate and compare the phenotypic characteristics (expression of MHC class II and costimulatory molecules) and functional T cell stimulatory and regulatory capacity of various CMS ARC populations (CNS-resident microglia, and CNS- infiltrating macrophages and DCs) for activation of both naive and activated (Th1/Th2 clones) myelin- specific T cells. These studies should enhance our understanding of the mechanisms of induction of epitope spreading and the effector role of these responses in the chronic pathogenesis of autoimmune diseases and provide information vital for the rational design of immunoregulatory strategies for their treatment. [unreadable] [unreadable] [unreadable]